Field of the Invention
The present invention is directed to glycan-specific analytical tools, their methods of use, and processes for making glycan-specific analytical tools. Other analytical tools are further provided herein.
Background Art
Glycans are complex carbohydrates commonly found attached to lipids and proteins. Because of their presence on protein and cell surfaces, complex carbohydrates often occupy a functional position in biological recognition processes. The complex shape, functionality, and dynamic properties of oligo- and polysaccharides allow these molecules to function in intermolecular interactions as encoders of biological information.
Carbohydrate recognition is an integral part of normal biological development, but can also be used by the innate immune system to allow a host organism to identify a foreign pathogen, on the basis of the carbohydrates presented on the surface of the pathogen. Conversely, many bacterial and viral pathogens initially adhere to host tissues by binding specifically to carbohydrates on the host's cell surfaces. Thus, there is an interest in developing therapeutic agents that can interfere with carbohydrate-based host-pathogen interactions or that can function as antibacterial vaccines. Abnormal glycosylation is also a marker for certain types of cancer and other diseases, making them targets for diagnostic and therapeutic applications. For example, the state of modification of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAcylation) is an important biomarker of changes caused by disease, notably type-2 diabetes mellitus.
Despite the importance of glycans in biological development and disease, there is at present a lack of sufficient glycan-specific analytical tools, which has delayed exploiting aberrant glycosylation in the diagnosis and treatment of disease. For example, a current method for monitoring O-GlcNAc incorporation in cells, and subsequent presentation on proteins, is based on exogenous uptake of labeling reagents, such as N-azidoacetylglucosamine (GlcNAz). Unfortunately, this method is not applicable to the analysis of O-GlcNAc in isolated tissue or protein samples. An alternative O-GlcNAc labeling approach that can be applied in glycomic/proteomic analyses uses chemoenzymatic tagging. A serious limitation of this method is that it also labels other GlcNAc-terminated complex glycans. Thus, there remains a need for analytical tools with defined carbohydrate specificity that can be used to interrogate biological samples in the search for abnormal glycosylation.
Currently, two major types of biomolecules used in glycan-specific analytical applications are sugar-binding proteins (lectins) and antibodies. A major drawback associated with either of these types of reagents is the characteristically weak interactions between carbohydrates and proteins, with dissociation constants typically in the range of milli- to micromolar for lectins and micro- to nanomolar for antibodies. Additionally, a significant difficulty in using antibodies is that carbohydrates are very poor immunogens. They are generally unable to generate a T-cell dependent response and so produce most often IgM class antibodies, which are inconvenient for analytical and diagnostic applications. Single chain chimeras consisting of the variable domains of the heavy and light chains (scFv) can suffer from instability. Additionally, glycan-specific analytical techniques employing antibodies suffer a drawback due to the selectivity of antibodies being context dependent. Alternatively, lectins, with their broad specificity, are limited in their use for analytical applications. Therefore, there exists a need for developing analytical reagents that possess sufficient specificities to the carbohydrate sequence, yet are able to recognize the sequence within a broad range of glycans.